This invention relates to a method for the analysis of chemicals in liquids. This is accomplished by means of a dry chemistry test device made up of one or more selective amperometric sensor elements coated with specific chemical reagent layers and an electronic apparatus that provides a pulse measurement voltage to the sensors.
The analysis of chemical species in liquids can be carried out by wet chemistry or by dry chemistry systems. Wet chemistry analyses employ reagents in liquid solution and are widely used in both manual and automated analytical methods. In dry chemistry systems, a complete chemistry for a particular analysis is miniaturized into a single test probe or device. No prior reconstitution of reagents is required. The dry chemistry methods are often simpler in design, require less reagent manipulation, give quicker results and are more stable. These advantages coupled to the feasibility of using small instrumentation favours more decentralized clinical testing.
In a U.S. Pat. No. 4,042,335 issues to Clement on Aug. 16, 1977, a multilayered test device for the analysis of liquids is described. The device includes a reaction layer including a composition that is interactive in the presence of a predetermined analyte to provide a diffusable detectable species, and a registration layer that is permeable to the detectable species and within which the species can be detected by diffuse reflectance spectroscopy. The element can include other layers which provide for radiation blocking and for uniform spreading of the test sample.
Dry chemistry systems can be defined as disposable, single-use devices or multi-use devices, depending on whether the analytical reaction occurring in the reaction layer is reversible.
Dry chemistry tests based on enzyme-catalysed reactions have become widely used. By way of example are dry chemistry test devices for the measurement of glucose in blood developed by Boehringer Mannheim Corporation. The device is a flat strip that employs a reaction layer consisting of glucose oxidase-peroxidase enzymes coupled to a redox mediator dye. The reaction layer is supported in a inert film carrier matrix. The blood sample is analysed by placing about 50 ul of whole blood on the surface of the dry chemistry strip. The plasma containing the glucose is separated from the red blood cells by the carrier matrix. After about 1-3 minutes, the red blood cells are removed by washing or wiping and the colour developed is measured by reflectance spectroscopy. In a similar device developed by Ames Division, Miles Laboratories Inc., a separate film membrane is placed over the carrier matrix to separate the red blood cells.
Electrochemical dry chemistry enzyme test devices have been developed as an alternative to spectrophotometric devices. These include amperometric and potentiometric devices. Electrochemical systems have the advantage that they can be used as disposable, single-use devices as well as multiple -use devices in continuous-flow and automated systems.
Amperometric dry chemistry enzyme test devices include a sensing element comprising a measuring or working electrode and reference electrode. The electrodes are coated with an enzyme and optional mediator and, over this, a membrane to prevent interfering species from reacting at the measuring electrode. When an appropriate test potential is applied, the measuring electrode provides a faradaic current proportional to the concentration of the chemical species being determined.
As an example of prior art in this field, U.S. Pat. No. 3,539,455 issued to Clark in Nov. 1970 discloses an amperometric dry chemistry device based on glucose oxidase suitable for the determination of glucose.
The role of the redox mediator is to serve as an electron shuttle between the active center of the enzyme and the electrode. Various redox mediators have been used in enzyme electrodes. For example, an article published in Clinica Chimica Acta in 1974 describes a range of mediators which include ferricyanide, quinones and organic dyes. European patent application EP 0,136,362,A1 published on 04 Oct. 1985 describes an amperometric test device for monitoring blood-glucose levels comprising a measuring electrode and reference electrode coated with a porous support carrying glucose oxidase and a redox mediator, and over this, a membrane to prevent interfering molecules from reacting at the measuring electrode.
Higgins et. al. have described the use of ferrocenes as a mediator for glucose oxidase-based enzyme sensor for glucose in European patent application EP 0,127,958,A2 published on Dec. 12, 1984; U.S. Pat. No. 4,545,382 dated Oct. 8, 1985; and U.S. Pat. No. 4,711,245 dated Dec. 8, 1987. Another mediator system employs organic metal coatings based on salts of tetracyanoqunidimethane as first described by Cenas and Kulys, Bioelectrochem Bionerg. 8(1981)103 Albery has filed an application based on this work in European patent application EP 0,184,909,A2. Albery et. al. have also shown that tetrathiafulvalene can act as a mediator for glucose oxidase in the determination of glucose ( W.J. Albery, P.N. Bartlett and D.H. Craston, J. Electroanalytical Chemistry, 194(1985)223).
Although spectrophotometric and amperometric dry chemistry test devices are gaining increasing usage, present systems suffer from a number of practical limitations.
First, in the case of disposable single-use dry chemistry test devices, it is not possible to calibrate every measurement. Presently the practice is only to calibrate every batch by means of a two point calibration using test devices representative of the batch. It is then assumed that the calibration holds for the remaining devices in the batch. The difficulty that arises is that there is no way of knowing if any of the remaining devices are defective for whatever reason.
A further consideration is that, because the measurement of chemical species in blood is sensitive to the blood matrix, it is necessary to use reference calibrators that mimic the average human blood matrix as close as possible. The need for calibration is even greater for amperometric devices where changes in the electrode response characteristics can occur for a number of reasons.
Further, single use test devices, especially for blood-glucose monitoring are often intended for home care applications where the user is generally not highly skilled or trained. Consequently, poor analytical results may be obtained where the user technique is incorrect or where the test devices have been badly handled or stored in adverse conditions.
Second, present disposable, single-use test devices normally can determine only one chemical species. Thus, if several different analyte in a particular sample have to be determined, multiple samples have to be obtained and a different sensor must be used used for each analyte. This is time consuming, requires extra manipulation and is expensive.
Third, in present systems because of the requirement that all devices in a batch must be identical, the manufacturing and quality control process must be quite stringent increasing the cost of production. Again this is more difficult to achieve for dry chemistry devices based on amperometric sensing.
Fourth, redox mediators that are commonly used in amperometric glucose-oxidase based sensors have increased solubility in the active, oxidised form. Thus, when the sensor is continuously poised at a positive operating potential in multiple-use applications, loss of mediator in the oxidised form occurs readily from the electrode surface. This leads to a loss of sensitivity and a limitation in the linear range with time.
Fifth, even though the use of a redox mediator enables the operating potential to be reduced by several hundred millivolts, significant interference from electroactive species such as ascorbic acid is still found. For some cases the mediator can actually enhance the signal due to the interfering species. This leads to erroneous results that can have serious clinical consequences.
Because of these problems, previous approaches to enhancing the performance of amperometric dry chemistry enzyme test devices have not been entirely successful.
Accordingly, it is an object of the present invention to provide a general amperometric dry chemistry enzyme test device for measuring chemical species such as glucose and cholesterol in liquids.
It is another object of the present invention to provide a method for measuring glucose concentrations in liquids using the enzyme glucose oxidase.
It is a further object of the present invention to provide a method for measuring chemical species such as glucose and cholesterol in liquids by providing a means for calibrating the test device for each measurement.
Yet another object of the invention is to provide a method for measuring chemical species such as glucose and cholesterol in liquids using a test device having multiple sensors to simultaneously measure more than one chemical species.
Still, a further object of the present invention is to provide a method for measuring chemical species such as glucose and cholesterol in liquids using a selective sensor element comprising an electrode system coated with an perfluorinated cation exchange polymer film incorporating a redox mediator.
Yet another object of the present invention is to provide a method for measuring chemical species such as glucose and cholesterol in liquids using a pulse detection mode to minimise loss of the mediator and enable reactivation of the sensor electrode.
Still other objects of the invention will in part be obvious, and will, in part, be apparent from the following description.